The present invention relates generally to a method and system of preparing specimens for use in transmission electron microscopy (TEM). More specifically, the present invention relates to a method and system for automatically preparing TEM samples for examination by depositing extremely small samples onto a grid without need for a blotting step.
Transmission electron microscopy (TEM) is a microscopy technique whereby a beam of electrons is transmitted through an ultra-thin specimen such that the electron beam interacts with the specimen as it passes through. The interaction of the electrons transmitted through the specimen in turn creates an image. This created image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or is detected by a sensor such as a CCD camera. The benefit is that TEM systems are capable of imaging at a significantly higher resolution than traditional light microscopes due to the small wavelength of the electron beams. This enables the instrument's user to examine fine detail, such as a single column of atoms, which is tens of thousands times smaller than the smallest resolvable object in a light microscope. TEM forms a major analysis method in a range of scientific fields, in both physical and biological sciences.
TEM specimens must be prepared and placed into gridded specimen holders to allow for insertion of the specimen holder into a vacuum column. The sample is placed onto the inner meshed area of the grid. Usual grid materials are copper, molybdenum, gold or platinum. This grid is placed into the sample holder which is paired with the specimen stage. A wide variety of designs of stages and holders exist, depending upon the type of experiment being performed.
The principal difficulty in the prior art is that sample preparation in TEM can be a complex procedure. TEM specimens are required to be at most hundreds of nanometers thick because the electron beam interacts readily with the sample, an effect that increases roughly with atomic number squared. High quality samples will have a thickness that is comparable to the mean free path of the electrons that travel through the samples, which may be only a few tens of nanometers. Preparation of TEM specimens is specific to the material under analysis and the desired information to obtain from the specimen. As such, many generic techniques have been used for the preparation of the required thin sections.
Materials that have dimensions small enough to be electron transparent, such as powders or nanotubes, can be quickly prepared by the deposition of a dilute sample containing the specimen onto support grids or films. The difficulty is that the deposition of these solutions generally results in the creation of a droplet that is too large and thick for sampling requiring that the sample be blotted. This is accomplished through the use of filter paper which introduces high shear to the specimen and is done at the expense of a great deal of time, both of which limit the ability to examine certain specimens and the formation of certain natural structures over time.
Once the specimen is deposited, in the biological sciences in order to withstand the instrument vacuum and facilitate handling, biological specimens can be fixated using either a negative staining material such as uranyl acetate or by plastic embedding. Alternately samples may be held at liquid nitrogen temperatures after embedding in vitreous ice. The biological material is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid freezing, usually in liquid ethane near liquid nitrogen temperature. By maintaining specimens at liquid nitrogen temperature or colder, they can be introduced into the high-vacuum of the electron microscope column.
There is therefore a need for an automated system for the preparation of a TEM sample. There is a further need for an automated system that automatically places and controls the size of a sample droplet formed in a TEM sample while eliminating the need for a blotting step. There is a further need for an automated system that automatically places and controls the size of a sample droplet formed in a TEM sample while eliminating the need for a blotting step in a manner that accelerates the preparation process thereby allowing previously unobserved structures to be viewed.